On May 27, GlaxoSmithKline (GSK) announced the European approval of the first ex-vivo stem cell gene therapy to treat patients with ADA-
SCID, a severe immunodeficiency resulting from the missing enzyme adenine deaminase. And although the condition is extremely rare—only about 15 new
cases appear each year in Europe—the announcement was viewed as a significant milestone.

“This is the
start of a new chapter in the treatment of rare genetic diseases, and we hope that this therapeutic approach could also be used to help patients with other
rare diseases in the future,” offered Martin Andrews, head of GSK’s Rare Disease Unit, in announcing the approval.

Stimvelis, as it has been commercialized, involves the isolation of CD34+ hematopoietic stem cells (HSCs) from ADA-SCID
patients, and transducing the HSCs with a retrovirus that carries the normal ADA gene. With a single administration, patients begin to produce ADA,
eliminating the need for enzyme replacement therapy—and, because the cells came from the patient, there is no risk for immune rejection.

The announcement is part of a broader renaissance in the field of gene therapy and, in particular, the synergies that are
developing between that field and the world of stem cells.

Viruses and
more &nb
sp;

There are a variety of mechanisms for getting normal copies of genes into cells. On the viral vector side, two of the most
common systems are adeno-associated virus (AAV) and lentivirus, which Daniel C. Smith, chief scientific officer for Cobra Biologics, suggests are currently broadly used for different gene therapy applications.

“AAV vectors are being predominantly used for direct gene therapy interventions that target disease in specific tissues through the selection of
specific natural serotypes and increasingly through the generation of novel hybrid serotypes,” he explains.

AAV is not associated with any human disease, he continues, and has low immunogenicity due to its small size. Thus, it has the potential of being used
for prolonged courses of treatment before the patient’s immune system inactivates the virus, decreasing its efficacy.

Clinical interest for AAV-mediated gene therapies, he suggests, has been encouraged by:

Improved understanding of
AAV serotype tropisms

Identification of serotypes with high transducing efficiencies, (i.e., AAV9)

One of the challenges of working with AAV vectors, suggests Thomas Barnes,
chief scientific officer of Intellia Therapeutics, is that because the virus cannot replicate,
the introduced gene is slowly diluted as the cells divide in the patient, meaning continued dosing.

“The
therapeutic gene delivered from the lentiviral vectors can permanently integrate into the patient’s chromosome, which could provide lifelong
therapeutic treatment from a single treatment,” Smith explains, adding that these vectors are predominately used ex vivo to engineer immune
cells.

The potential benefits of integration have been tempered somewhat by concerns over whether such events
would lead to new problems, such as the disruption of vital genes or activation of oncogenes.

According to Louis
Breton, CEO of Calimmune, this really hasn’t been an issue.

“What’s nice is that there are literally thousands of man-years of data now, because there have been hundreds of patients treated with
lentiviral vectors, specifically,” he says. “Integration site analysis has been done by lots and lots of groups to provide additional data on
previous concerns about the use of lenti.”

“And beyond the safety profile, the whole reason why people
were using them is because of the efficiency of delivering payload,” he continues. “A primary element of making sure that the therapy works is
making sure you can get the therapy into enough cells to make a difference.”

For Calimmune, that means using
these vectors to knock down or disrupt specific cellular functions (more below), while for others, it may mean increased production of a missing metabolic
component.

Viral vectors are not the only way to introduce normal genes into diseased tissue, however. There is a
growing market of integrating, long-term expressing vectors based on transposons, with names like piggyBac (PB) and Sleeping Beauty (SB).

Publishing in Cancer Gene
Therapy last year, L.J.N. Cooper and colleagues at MD Anderson Cancer Center demonstrated that they could use the SB platform to generate CD19-
specific CAR T cells in cGMP compliance as part of a broader first-in-human clinical program. The authors suggested that the SB system offered the advantages
of cost and simplicity of naked DNA with the gene transfer efficiency of recombinant retroviruses.

John Wolfe and
colleagues at Children’s Hospital of Philadelphia, meanwhile, used PB transposons to try to reverse the neuropathology associated with lysosomal
storage diseases. As they described in Stem Cell Reports, the
researchers reprogrammed fibroblasts from human patients with Sly syndrome into neuronal stem cells (NSCs) via an iPSC intermediate. They then genetically
corrected the cells with a PB transposon carrying the GUSB gene.

Upon injecting these cells into mouse brain, they
noted that not only did the NSCs engraft around the injection site, but they could also detect GUSB enzymatic activity as well as immunological and
morphological signs of lesion healing.

Liguo Luo and colleagues recently reviewed PB transposons, arguing that unlike many viral vectors and even
other transposons, PB can accommodate much larger genetic payloads while offering what they described as footprint-free transposition, whereby the transposon
does not alter the DNA sequence when it moves.

For its part, Intellia has focused its efforts on gene editing
rather than gene therapy via insertion. (See also “Germlines and gene-
editing” in the August 2015 issue of DDNews.)

“I think gene therapy and gene editing are
complementary,” Barnes says. “Gene therapy is good to replace functions that are missing in people, but it’s not so good at removing
functions that are gumming up the works, if you will.”

“Where you have dominant disorders, [gene]
knockout would be advantageous,” he continues. “It’s something that CRISPR can do very well.”

And, he continues, if you are interested in repairing genetic defects, you don’t necessarily have to provide the whole gene, but rather you may
be able to repair part of the gene with something like CRISPR/Cas9. And unlike AAVs, because gene editing modifies the chromosome, the effect remains with
the cell and its descendants.

Looking to leverage gene editing and viral vectors in immuno-oncology as well as
autoimmune and inflammatory disease, Intellia recently launched a new division dedicated to ex-vivo approaches—eXtellia Therapeutics. The
launch came a full year after the company had entered a collaboration with Novartis to explore ex-vivo technologies for the latter’s CAR T
cell pipeline and its efforts with HSCs.

“When the immune system is a little bit asleep on the job,
it’s going to let through some cancer cells and eventually, they’re going to take hold and make a tumor,” says Barnes. “But the
system can go awry in the opposite direction. It can be a little bit overactive and start finding cells that should be there and attacking normal tissue.
That is autoimmune disease.”

“The beauty of cell-based therapy over traditional drugs is it is a
living drug; it replicates,” he presses. “So you can reintroduce cells into a patient, and they can have an enduring response to cancer or you
can re-establish tolerance to the tissue that’s being attacked, and have that maintained and sustained.”

“The therapeutic applications of genome editing that are closest to clinical translation are disruption of the HIV-1 coreceptor CCR5 to treat
HIV and of the γ-globin repressor BCL11A as a therapy for β-globinopathies,” offered Paul Cannon and colleagues at Sangamo BioSciences and the University of Southern California. “Both of these programs involve gene
knockout, whereas the ability to correct mutations or add DNA sequences would substantially broaden the impact of gene-editing technologies.”

As they described in Nature Biotechnology last year, the researchers were also interested in applying gene-
editing technologies to stem cells, but in their case, they delivered ZFN mRNA and AAV6-mediated template into CD34+ hematopoietic stem and progenitor cells
(HSPCs). They found that not only could they efficiently modify the HSPCs, but also that these cells engrafted long term in immune-deficient mice, suggesting
the cells maintained their stemness.

“We’re taking a patient’s own cells, treating them and
giving them back,” Breton explains, “so there are a number of different questions that you do have to check the box for.”

“Not just whether the cell remains a stem cell through the processing, but when you put it back, does it actually do
what it’s supposed to do?” he continues. “Does it act like a normal HSC would? Does it differentiate? Are the lineage of cells that it
produces similar?”

“You want to make sure that the therapy that you are using is not affecting the
machinery of the cell to become the important lineage cells necessary to be the warrior cells of the body and ultimately protect the patient,” Breton
concludes.

“Fortunately, there are a lot of differentiation assays that people use to assess whether cells
can differentiate into the major types of lineages,” adds Barnes, including not only in-vitro tests for HSCs, but also in-vivo
tests.

As Barnes explains, one can transplant a cell into, say, a mouse, and if it has stem cell properties, it
should be able to completely repopulate the blood. And, you should also be able to generate a cell that can be taken from one mouse and be used to repopulate
another mouse in a process called serial transfer.

But as Breton is quick to remind us, animals are not humans.
How are these technologies being moved to the clinic?

Viruses tackling viruses

In something of a biological irony, Calimmune is actually using lentiviral vectors to offer gene-therapy treatments to
patients with HIV, itself a lentivirus.

According to Breton, HIV occurs in one of three tropic forms. The major
form latches onto chemokine receptor CCR5 to enter the cell. Thus, patients who are homozygous negative for CCR5 or in whom CCR5 has been inhibited are
effectively immune to HIV infection (see sidebar article From Boston to Berlin sidebar article below after the end of this main article).

A less-frequent form is tropic for the CXCR4 co-receptor, which the virus uses to fuse with the cell membrane. And finally,
there is a form that can use potentially both co-receptors.

It is because of these latter two tropic forms that
Calimmune decided to target both entry mechanisms with its Cal-1 program.

As Breton describes, Cal-1 is a
lentiviral vector that includes both an shRNA to down-regulate CCR5 expression and the fusion inhibitor C46, which interferes with CXCR4 tropism.

As Calimmune’s Geoff Symonds and colleagues noted in a preclinical study published in 2014: “LVsh5/C46-treated
CD34+ cells maintained their ability to differentiate into various hematopoietic lineages with no sign of lineage skewing. Moreover, LVsh5/C46-modified cells
showed profound resistance to R5-, X4-, and dual-tropic strains of HIV-1.”

“We are now moving through
the Phase 1/2 clinical study, and we are in the final cohort of patients in a dose-escalation of conditioning,” Breton continues. “Conditioning
allows these cells that you give back to go into the marrow and actually contribute for long-term lineage of protected cells for the patient.”

One challenge with inserting modified stem cells into the body, however, is figuring out how to ensure the new cells
replace the errant cells already in place. In some ways, Calimmune has an advantage over many other companies.

“What’s unique about doing gene therapy in HIV is that there is a natural selection process,” Breton enthuses. “The cells that
are protected would be able to grow; those that are not, HIV would target directly and eventually kill.”

“In other disease areas that are looking at ex-vivo autologous cell therapy—and gene-based therapy specifically—it is a big
question.”

But he is enthusiastic that those questions will soon have answers.

“A lot of the work in the basic cell science—as well as understanding which cells propagate, which ones will
hone, targeting of cells—has required technological advancements in order to have some of the breakthroughs that are starting to emerge in some of
these other disease states,” he chimes. “It’s actually quite exciting because there’s a lot that has been accelerated learning per se
in the field over the course of the last five years.”

In a Phase 1 study being conducted by Toronto’s
University Health Network (NCT02800070), researchers are looking at a first-in-human study for the treatment of Fabry disease, relying on autologous stem
cell transplantation using CD34+ cells that are transduced with the lentivirus vector containing the human alpha-gal A gene. The study, which only recently
started recruiting, will examine whether re-introduction of transduced cells will help increase alpha-gal A enzyme levels as well as transplantation safety
and toxicity.

Similarly, in a Phase 1/2 study led by UCLA’s Donald Kohn (NCT01852071), up to 20 infants and
children diagnosed with ADA-deficient SCID will receive autologous transplant of CD34+ bone marrow stem/progenitor cells transduced with a lentiviral vector
carrying the human ADA gene. The primary focus of the study is safety, but follow-up analysis will determine whether the cells can engraft and produce mature
cells that contain and express the corrected ADA gene in the absence of PEG-ADA enzyme replacement therapy.

Meanwhile, bluebird bio is using lentivirus-transduced CD34+ stem cells in a Phase 1/2
study of its LentiGlobin BB305 program in patients with beta-thalassemia. Reporting on the Northstar Study at ASH in December, Mark Walters of UCSF Benioff
Children’s Hospital suggested that all of the subjects treated with the βA-T87Q-globin had experienced sufficient clinical benefit to
be transfusion independent (non-β0/β0 genotype) or experience transfusion volume reduction
(β0/β0 genotype).

Having the end in mind

Unlike the small-molecule world where scaling up can be as simple as larger amounts of starting material, transitioning a
gene- or cell-therapy project from the preclinical to clinical space can be significantly more challenging.

“I think that there’s a lot to be said about making sure something is effective before it becomes efficient,” says Breton, who
cautions about jumping too far ahead in one’s thinking about therapeutics. “A lot of the smaller-scale work that’s been done has been very
good.”

Building those efforts to a larger scale is important, however, and he believes that having the end
in mind is a critical step.

“At Calimmune, that is one of the things that we have really focused on, knowing
that there are a number of these types of obstacles to ultimately bringing this to larger scale patient segments,” he continues. “However, it is
important to say that a lot of the work that is now within companies really was formed, developed and ultimately carried out early in a lot of these academic
centers that have ultimately been the breeding ground for great science.”

For Cobra’s Smith, ultimate
GMP compliance has to be designed into the product and the intended manufacturing process, including the raw materials, starting materials and consumables
used to produce the product from the start. And in Cobra’s case, those considerations start with the customer.

“As with all projects, there is a need to work with customers to ensure that the required design concepts have been built into the product,
including the vector design, and then working with them to develop compliant production processes,” he says.

Smith suggests the real challenges with these products are the inherent complexity of the products being produced, the associated supply chain for
production, and what he sees as a lack of robust and orthogonal analytical tools to interrogate the manufacturing process or resulting products.

Simply put, the process analytical technologies characteristic of chemical production lines are much less mature and in some
cases, vastly more complicated for the biological lines.

There is a greater effort, recently, to address these
challenges, says Breton.

“What’s nice today is that there are more companies and obviously more
capital that has come into gene therapy that is allowing the companies to be able to leapfrog a lot of the work that had been done and continue to move
toward next-generation,” he enthuses. “Most of the academic institutions were not necessarily looking to commercialize product, but bring it as
far as they could in order to make it a feasible potential path. And we see that as a very important relationship with industry and one that we have
certainly cultivated from our roots, as well.”

He points to Calimmune’s Cytegrity platform that is set
up to provide durable and scalable manufacturing in large quantities for therapeutic GMP production.

“What
that really means is being able to produce in batch sizes that are much further beyond the academic and small-batch brews that many groups have been
conducting through previous transient transfection means,” he explains. “Where people were doing this in flasks before, we’re looking at
larger bioreactors that would allow for there to be scale. And that is part of the process.”

And, like
discovery-phase research, production technology development is constantly evolving, particularly when looking to meld stem cells with gene therapy.

In March, GSK and Miltenyi Biotec announced a strategic
collaboration to explore synergies between the former’s expertise in cell- and gene-based therapy and the latter’s in cell processing
technologies and automation. In particular, the companies will work to advance the discovery and development of CAR T-cell therapeutics, enhancing
GSK’s existing preclinical pipeline.

Similarly, bluebird bio is looking ahead as its clinical programs
progress and recently announced a long-term commercial manufacturing agreement for its Lenti-D and LentiGlobin programs with Lonza.

“As we advance our gene therapy programs through clinical trials, we are deliberately building key infrastructure and
relationships in preparation for commercial launch,” said chief bluebird Nick Leschly in announcing the agreement. “Our partnership with Lonza is
one notable example of our progress on the manufacturing front, and we are pleased to benefit from their expertise and experience.”

“The collaboration with Touchlight creates the opportunity to link highly innovative technologies in the manufacture of
DNA vectors to the production of gene therapy viral vectors, thus creating the potential to reduce timelines and cost for the production of those viral
vectors,” Smith explains. “These are critical issues that need to be addressed if these therapeutic approaches are to be
commercialized.”

The company will evaluate the integration of Touchlight’s synthetic doggyboneDNA and
Cobra’s antibiotic-free plasmid DNA system for reductions in timelines and costs for AAV vector production.

The collaboration with CPI, meanwhile, will initially focus on the development of methods suitable for AAV vector characterization and in-process
analytics.

Such efforts are about more than just costs, however, according to Breton; it is also ultimately about
product quality.

“You want to have the highest-quality therapeutic possible, and something that allows you
to have quality, and these types of vectors will ultimately have an impact on everything from transduction efficiency to the ability obviously to have a
therapeutic impact for the patients,” he offers.

It’s about being able to set a standard where you can
have batch-to-batch consistency on an ongoing basis with cell lines that are stable for generations.

“This
is a really important part of what we believe to be connected to the clinical development and ultimately the commercial requirements for gene therapy,”
Breton suggests.

Smith suggests that we will be surprised by how quickly these goals become a reality.

“Whilst there are still very significant technical hurdles to address, it is clear that a number of highly capable
scientists and engineers are now entering the field backed by significant funding, and there is real opportunity the potential of gene therapy to be finally
realized,” he says.

“New advanced production systems will lead to significant increases in
productivity, decreasing the cost of goods for these therapies,” Smith continues. “This will lead to greater opportunities for the field as it
will become financially viable to manufacture for larger indications.”

The enthusiasm is infectious.

Taking their place

“More and more, the CAR T cell
work has gotten a lot of visibility, which I think is great,” opines Breton. “But there is a lot of work that has been done and continues to be
done with the use of HSCs; and in our case, it’s ex-vivo autologous cell therapy. There is now a tremendous amount of data that is starting to
actually show evidence that these therapies do have the capabilities of having lasting impact.”

“If
you look back four or five years ago, there just wasn’t as much effort, capital, even interest at being able to move down these paths,” he
remembers, contrasting that with today when the clinicians and scientists that worked literally decades to move some of these programs along are starting to
see the fruits of their labors provide benefits to patients.

“The field has changed enormously over the last
few years with the influx of significant funding especially into the immunotherapy area,” echoes Smith, “and will continue to change as an
increased number of pipeline of products are brought to the clinic and existing products progress through into later stage clinical studies and hopefully
onto the market.”

“It’s still early for a number of programs,” Breton cautions, “but
the signs are looking promising.”

From Boston to
Berlin

In 2007, an HIV+ patient who had developed leukemia was given an allogeneic bone marrow transplant
(BMT) in an attempt to combat his cancer. The treatment was successful not just in reversing the “Berlin patient’s” leukemia, but also
reduced his HIV load to undetectable levels, even after he ceased his antiretroviral therapy (ART). The BMT essentially cured his infection.

This was not, however, the first time such an outcome had been seen. Earlier, two HIV+ patients in Boston had similarly seen
their viral loads disappear upon receiving BMT for cancer. But unlike the Berlin situation, months after the Boston patients were taken off ART, the HIV
levels increased again. They were not cured of HIV infection.

Gero Hütter, treating physician for the Berlin
patient, offered his thoughts in a recent editorial in Expert Review of
Hematology.

“Despite the fact that the patients seem to be virus-free, both rebounded from HIV after
the antiretroviral medication was discontinued,” he wrote. “Interestingly, in these patients, the rebound appeared after several months (instead
of days), indicating that the allogeneic SCT [stem cell transplant] and concomitant immunosuppression may have reduced the size of the reservoir but in the
end was not powerful enough to eliminate the virus completely.”

In an unanticipated
coincidence, the individual who had donated bone marrow to the Berlin patient was homozygous for a mutated form of the chemokine receptor CCR5 (CCR5-d32), a
cell-surface protein that HIV relies upon to invade human cells. The effort was on to repeat these results in other HIV+ cancer patients.

Unfortunately, as Hütter recounted, “Although several attempts have been undertaken to repeat this approach, most
published cases were not evaluable because patients receiving the CCR5-deficient stem cells died from procedure side effects or cancer relapse soon after
transplantation.”

Another major problem is that the global incidence of people homozygous for CCR5-d32 is
about 1 percent, and even if you could harvest cells from each of these potential donors, HLA matching would still limit the ability of getting these cells
into recipients.

That said, many companies have started to focus on ways to disrupt the interaction of HIV with
CCR5, whether with small molecules like Pfizer’s maraviroc or through molecular interventions such as Calimmune’s shRNA construct or
Sangamo’s ZFN gene-editing efforts.

Further complicating the CCR5-directed approach, however, are the
results with another patient who received homozygous CCR5-d32 SCT. Although the “Essen patient” initially experienced a drop in viral load, the
levels soon rebounded with a form of HIV tropic for the CXCR4 co-receptor, which facilitates the fusion of HIV particles with cell membranes.

Monotherapy targeting of CCR5 may, therefore, be insufficient to reduce or eliminate HIV infection. That why Calimmune has
decided to target both mechanisms in its Cal-1 clinical program, explains company CEO Louis Breton. The program is in the late stages of a Phase 1/2 clinical
study.

First strike vs. last resort

With
some health conditions like inborn errors of metabolism, the lack of a protein or other factors can start having deleterious effects the moment the infant is
born, when it can no longer rely on its mother’s body. Rather than subject the child to a lifetime of replacement therapy, however, some researchers
are questioning whether the genetic error cannot be fixed in utero.

In a recent issue of Molecular
Therapy—Methods & Clinical Development, Christopher Porada and colleagues at Wake Forest Institute for Regenerative Medicine discussed the current state of in-utero gene therapy (IUGT) and its potential
evolution from preclinical challenge to clinical reality. They centered their discussion on the treatment of hemophilia A (HA), where patients largely lack
clotting factor VIII (FVIII).

Rationale: Despite the success of protein-replacement therapy, a
lifetime of weekly infusions at a cost of upward of $300,000/year is a significant burden on patients. As well, almost a third of patients who can access
treatment will mount an immune response to FVIII, diminishing or even eliminating treatment efficacy.

Opportunity: For most cases, prenatal diagnosis is not only feasible, but can be done with little risk using digital PCR of fetal
cells released into the peripheral blood of the mother. (See also “Non-invasion of the body snatchers” in the October 2015 issue of DDNews.)

Furthermore, the
authors suggested cells within the fetus are much more active and amenable to transduction with viral vectors than similar cells in adults. They cited their
efforts in sheep and others that showed they could achieve gestational gene transfer levels in hematopoietic systems of 5 to 6 percent, enough to convert
severe HA to a moderate or even mild phenotype.

“Even if the initial gene transfer only transduces a small
number of the desired target cells, this subsequent expansion could produce clinically useful levels of gene-correction by birth,” they wrote.

Fetal expression also has the potential to decrease concerns over immune response to FVIII later in life, they argued,
further improving a patient’s chances should later, post-gestational rounds of protein- or gene-therapy be indicated.

Risks: IUGT is not without its potential challenges, however, with risks to both the fetus and the mother. Although there is
little risk to the fetus from the procedure itself, more study is required to ensure that fetal development continues normally upon injection and that there
is no inherent toxicity from the vector. As well, one study has suggested that AAV vectors can cross the blood-placental barrier, which could lead to
transduction of maternal tissues.

There is also concern over the potential for integration-related insertional
mutagenesis, where the vector disrupts the function of a host gene. Although the authors were quick to note examples of where this has been a problem in the
past, they also highlighted their long-term sheep studies that showed no evidence of such issues over a lifespan equivalent to 35 human years.

“Preclinical assessment of the risk of insertional mutagenesis following IUGT will require very carefully designed
studies with the actual vector to be employed for the pending clinical trial, in an animal model that has been thoroughly validated in the setting of the
target disease.”

Reasons to hope: The authors quickly acknowledged that despite the great
strides that have been made in preclinical models, there is still a lot of work to be done and hurdles to overcome before IUGT can enter the clinic. By the
same token, they are quite hopeful.

“From our findings in the sheep model and those of other groups
exploring IUGT in sheep, mice, and nonhuman primates, it is clear that the direct injection of viral vectors into the developing fetus can be an effective
way of delivering an exogenous gene and achieving long-term expression in multiple tissues, suggesting that IUGT may one day be a viable therapeutic option
for diseases affecting any of the major organ systems,” the authors concluded.

“Moreover, even if not
curative, IUGT would be ideal for a disease like HA, since lifelong immunologic tolerance could be induced to FVIII, thus overcoming the immune-related
hurdles that currently hinder postnatal treatment of this disease,” they continued. “There is no doubt that surpassing the few remaining hurdles
to allow clinical implementation of these therapies will dramatically change the whole paradigm for the way we perceive and treat many genetic
disorders.”

NEW YORK—While steady progress in clinical research and increasing evidence
of product effectiveness are significant driving factors for growth in the stem cell therapies market, a number of obstacles remain before they become widely
commercially viable, according to business intelligence provider GBI Research.

One of the company’s latest
reports states that the stem cell therapy pipeline as a whole is relatively large, with 330 products in active development across all stages. Indeed,
research and development within the sector is gaining momentum as candidates move into clinical development and the results of clinical studies become
apparent, clarifying the therapeutic potential of stem cell therapy.

“The stem cell space provides
therapeutic potential in indications where current pharmacological and surgical treatment options are ineffective,” noted Rodrigo Gutierrez Gamboa,
managing analyst for GBI Research. “However, there remains a significant divide between the number of stem cell therapeutic applications currently
available for patients and the number of research programs investigating the wider medical applications of stem cell-based therapies.”

According to Gutierrez Gamboa, stem cells are only in use for a “modest variety of indications” right now,
pointing to skin and blood stem cells as representing most therapeutic uses.

“The stem cell industry is
pursuing a broad base of therapeutic applications, and this is evident in the R&D efforts observed in the sector, as over 15 therapy areas are being
targeted by the stem cell industry in over 1,000 clinical trials,” he said.

Despite promising recent
developments within the field of stem cell technology, converting its scientific potential into real therapeutic value still represents a significant
challenge. According to GBI Research’s survey of key opinion leaders, the stem cells field is still surrounded by a wide variety of obstacles, most
notably the high cost of research, which survey participants stated was the biggest factor limiting stem cell progress.

“Manufacturers will need to adopt novel strategies to realize their full potential. It is likely that manufacturing methodologies will use
partially or fully automated systems in future approaches, in order to improve yield, purity and cost-effectiveness,” concluded Gutierrez Gamboa.
“There have been good strides made by stem cell manufacturing companies, such as those made by Cynata Therapeutics, which is implementing innovative
manufacturing methods to generate robust, consistent and inexpensive stem cells. Such companies are pointing towards a promising outlook in this
regard.”

Stem cells improve blood vessel function following spinal cord
injury

DURHAM, N.C.—A new study published in STEM CELLS Translational Medicine
(SCTM) reportedly shows how a minimally invasive stem cell treatment in rats can reduce secondary damage in traumatic spinal cord injury (SCI).
While similar studies have also demonstrated the promise of stem cells as a therapy for SCI, what makes this one different, according to SCTM, is
the type of stem cell used. For the first time, researchers evaluated whether a brain-derived stromal cell would be better suited to target the acute phase
of SCI than cells derived from other tissue sources. The answer, apparently, was yes.

SCI is a life-threatening
condition with limited treatment options. It occurs in two phases. The primary phase takes place when the initial trauma causes mechanical injury to the
spinal cord. The secondary injury comes in the hours after. As the body attempts to deal with what has happened, it releases a surge of chemicals causing
inflammation, decreased spinal cord blood flow and cell death, which further exacerbates the injury. As the body cannot readily replace dying cells after
spinal cord injury, neurological function becomes permanently impaired, resulting in severe movement and sensory disabilities.

While studies in animals have shown that the transplantation of stem cells might aid spinal cord repair by, among other
things, replacing dead neural cells, the current study focused on suppressing damaging inflammation and improving blood vessel function, which may reduce the
extent of injury.

Central nervous system (CNS) pericytes (specialized cells surrounding the capillaries) have
recently gained significant attention within the scientific community. In addition to being recognized as major players in neural tissue trauma, pericytes
share a common origin and, potentially, a common function with traditionally defined mesenchymal stem cells (MSCs). Although these cells have been previously
studied in the lab, their therapeutic application in vivo has not been evaluated.

“Our study
demonstrates that these cells not only display a MSC phenotype in a dish, but also have similar immunomodulatory effects in animals after spinal cord injury
that are more potent than those of non-central nervous system tissue-derived cells. Therefore, these cells are of interest for therapeutic use in acute
spinal cord injury,” said lead investigator Dr. Michael Fehlings. The Fehlings research team, based at the Krembil Research Institute in Toronto
Western Hospital and the University at Toronto, conducted their study by injecting human CNS-derived stromal cells into rats with SCI, and compared the
results to a control group treated with MSCs. The cells protected blood vessels in the injured area, among other positive outcomes.

“These early effects further translated into enhanced functional recovery and tissue sparing 10 weeks after SCI,”
Fehlings added. “This work demonstrates a new therapeutic approach.”

LA
JOLLA, Calif.—Scientists at The Scripps Research Institute (TSRI) and Scripps Clinic have received a grant of nearly $2.4 million from the California
Institute for Regenerative Medicine (CIRM) to support safety and quality tests of a potential stem cell therapy for Parkinson’s disease.

The new two-year project will be led by Jeanne Loring, professor of developmental neurobiology at TSRI. Loring will be
partnering with Dr. Melissa Houser, neurologist and medical director of the Parkinson’s Disease and Movement Disorders Center at Scripps Clinic.

“The goal is to restore the quality of life for Parkinson’s patients,” said Loring. “The
methods we’re using will raise the bar for quality considerably—for all kinds of cell therapy.”

“What sets our study apart from many others is that it’s patient-specific,” said Houser. “Our hope is that this grant will
help to begin a new era of long-term treatment for Parkinson’s disease.”

Parkinson’s disease
strikes when specialized neurons in the brain begin dying. These neurons produce dopamine, a chemical messenger that maintains normal nerve-firing patterns.
Without dopamine, patients suffer from tremors, a lack of balance and even speech difficulties.

For the study, the
Loring lab will investigate induced pluripotent stem cells (iPSCs), which are derived from adult subjects and can differentiate into any kind of cell in the
body. In this case, iPSCs derived from cells donated by 10 Scripps Clinic Parkinson’s patients were developed into dopamine-producing neurons—the
same kind that die during Parkinson’s.

The new grant will allow the researchers to advance these cells
through U.S. Food and Drug Administration preclinical testing requirements, with the hope of moving closer to clinical trials.

Loring’s work focuses in part on improving the quality and safety of stem cell therapies. She and her colleagues
recently published the first comprehensive analysis of genomic sequence of iPSCs, and her lab’s advances include the development of the PluriTest
quality control assay for pluripotency (the ability of stem cells to differentiate), which was recently licensed to The Coriell Institute for Medical
Research.

Plasticell and National University of Ireland initiate cancer stem
cell collaboration

STEVENAGE, U.K.—On Aug. 1, Plasticell, a biotechnology company using
combinatorial technologies for stem cell research and the optimization of cell and gene therapy manufacturing, announced the signing of a collaboration
agreement with the National University of Ireland, Galway (NUI Galway) focused on methods of eradicating cancer stem cells.

Many malignant tumors are initiated and maintained by a discrete population of tumor cells that share many of the characteristics of normal adult
stem cells. However, unlike the majority of cancer cells that comprise a tumor, cancer stem cells (CSCs) are often refractory to chemotherapy and are thought
to be primarily responsible for relapse following cancer treatment. As the partners note, it has been shown that CSCs, appropriately stimulated by certain
drugs, become susceptible to chemotherapy, leading to complete tumor remission once drug treatment is withdrawn.

Plasticell’s proprietary Combinatorial Cell Culture (CombiCult) platform will be used to discover combinations of drugs, growth factors and
chemotherapeutic agents that are capable of eradicating CSCs in leukemia. The work will be carried out at Plasticell and in the laboratory of Dr. Eva
Szegezdi at the Apoptosis Research Centre of NUI Galway, and is partly funded by an Enterprise Partnership grant from the Irish Research Council.

“Despite much progress in the war against cancer, few definitive cures have emerged in the past 60
years,“ commented Dr. Yen Choo, Plasticell’s executive chairman. “However, research into cancer stem cells, together with recent
breakthroughs in cancer immunotherapy, seem set to provide a string of successes in the field. Plasticell is well positioned in both fields, being highly
experienced both in stem cell biology and in optimizing the manufacture of cell and gene therapies to bring advanced therapeutic medicinal products to
market.”

Pluristem gears up for Phase 3 trial of PLX-PAD in critical
limb ischemia

HAIFA, Israel—Pluristem Therapeutics Inc., a developer of placenta-based cell therapy
products, announced in early August that it had received positive feedback from the U.S. Food and Drug Administration (FDA) on the proposed Phase 3 trial of
its PLX-PAD cells in the treatment of critical limb ischemia (CLI)—a trial intended to support a biologics license application.

The PLX-PAD study is a double-blind, randomized, placebo-controlled trial in an estimated 250 patients with CLI Rutherford
Category 5 who are unsuitable candidates for revascularization. Patients will be treated with 300 million cells or placebo, injected twice intramuscularly,
with the second dose administered two months after the first. The primary endpoint will be time to amputation and death (amputation-free survival). Clinical
sites will enroll patients in the United States and Europe. In parallel, the study protocol has been submitted as a single pivotal trial to European national
competent authorities, following scientific advice from the European Medicines Agency (EMA), and approval is expected in the upcoming months.
Pluristem’s intention is to utilize this 250 patient trial as a single pivotal trial to apply for regulatory approval in both the United States and
Europe.

In CLI, fatty deposits block arteries in the leg, leading to greatly reduced blood flow, pain at rest,
non-healing ulcers and gangrene. Patients with CLI are at an immediate risk for limb amputation and death. With poor treatment options, CLI patients who
cannot undergo revascularization procedures have a high unmet medical need.

“This is a significant leap
forward for Pluristem, as we prepare to enter into a U.S. Phase 3 trial with our cell therapy for the treatment of CLI. There are few treatment options for
this serious cardiovascular condition, which too often leads to amputation and death. We look forward to starting this trial by early 2017,” stated
Pluristem Chairman and CEO Zami Aberman. “Concurrent with this U.S. FDA process, we are also moving the CLI indication forward in Europe and Japan. Our
PLX-PAD cells address a $12-billion global market in the treatment of CLI.”

As part of its global strategy,
Pluristem intends to conduct a pivotal trial in Japan in addition to the planned U.S. and European trials. Pluristem reached an agreement with Japan’s
Pharmaceuticals and Medical Devices Agency on the protocol of a pivotal trial in 75 patients for PLX-PAD in CLI via Japan’s accelerated regulatory
pathway for regenerative medicine. Pluristem’s strategic decision is to partner with a Japanese partner to conduct this study. In May 2015, the
EMA’s Adaptive Pathways Pilot Project selected the PLX-PAD program for the accelerated pathway, which may lead to conditional marketing approval
following a single successful pivotal study.

The design of the Phase 3 study protocol is based on two successful
Phase 1 trials in CLI. Patients in the Phase 1 studies were Rutherford categories 4 and 5, and not suitable candidates for leg revascularization. Data from
the two Phase 1 studies showed a favorable safety profile and promising data on amputation-free survival one-year post-treatment, improved tissue perfusion
and a reduction of ischemic pain at rest. An ongoing Phase 2 trial in intermittent claudication is expected to complete enrollment of its target of 170
patients by the end of 2016. Intermittent claudication is an earlier stage of peripheral artery disease that can precede CLI.